The allylic pyrophosphates, farnesyl geranyl pyrophosphate, are the universal acyclic procursors of sesquiterpenes and monoterpenes, respectively. Using a number of monoterpene and sesquiterpene synthetases isolated from fungi and higher plants, we plan to study the key cyclization reactions by which these widely occurring classes of isoprenoid natural products are biosynthesized. A proposed stereochemical model for the formation of six-membered ring-containing sesquiterpenes, involving the intermediacy of the tertiary allylic pyrophosphate nerolidyl pyrophosphate will be tested by the use of trichodiene synthetase and appropriately 3H- and 14C-labeled farnesyl and nerolidyl pyrophosphate substrates, allowing us to define the conformation of the cyclizing substrates as well as the role played by the pyrophosphate ion. The mechanism of the proposed isomerization--cyclization sequence leading to formation of trichodiene will be further investigated by comparing the mechanism of action of trichodiene synthetase with that of farnesyl pyrophosphate isomerase. The latter enzyme catalyzes the isomerization of farnesyl of nerolidyl pyrophosphate. By using a series of selected substrate analogs, we will attempt to factor the trichodiene synthetase reaction into its component transformations of isomerization and cyclization. We plan to study the stereochemical details of a second cyclization reaction, the enzymatic transformation of farnesyl pyrophosphate to the sequiterpene humulene, catalyzed by a cyclase isolated from sage. We have been testing a stereochemical model of monoterpene biosynthesis, using a variety of monoterpene synthetases. Incorporations of various labeled precursors, including linalyl pyrophosphate, are planned which will allow us to elucidate the detailed structure and conformation of the reactive intermediates of monoterpene biosynthesis. It is expected that the work proposed will lead to the development of new techniques for the study of natural products biosynthesis and that the information gained from these studies will be applicable to an understanding of the catalysis of biological carbon-carbon bond formation in general.